Abstract

2297

Squamous cell carcinomas (SCCs) of the head and neck are malignant tumors with high capacity to invade and metastasize. Matrix metalloproteinase-13 (MMP-13, collagenase-3) is specifically expressed in SCC cells, and its expression correlates to their invasion capacity. Transforming growth factor-beta (TGF-beta) enhances MMP-13 gene expression in SCC cells via p38 mitogen-activated protein kinase (MAPK). Here, we have elucidated TGF-∈2 signaling and the role of the Smad signaling pathway in regulating invasion proteinase expression in human cutaneous head and neck SCC cell line (UT-SCC-7), which expresses Smad2, Smad3, and Smad4 genes. In UT-SCC-7 cells, Smad2 was phosphorylated 15 min after TGF-beta treatment, and phospho-p38 MAPK was detected 120 min after TGF-beta treatment. Interestingly, basal phoshorylation of Smad3 was detected in UT-SCC-7 cells in the absence of TGF-beta, and it was further enhanced after a 30 min TGF-beta treatment. Basal phosphorylation of Smad3 was detected also in four other human head and neck SCC cell lines. In contrast, TGF-beta did not induce expression of CDK inhibitors p15, p21, and p27 in any of these cell lines. Adenovirally delivered HA-tagged Smad3 was also phosphorylated and translocated into the nucleus of UT-SCC-7 without TGF-beta treatment. Adenoviral overexpression of Smad3 enhanced the effect of TGF-beta on MMP-13 mRNA and protein levels by 2-fold, while dominant negative Smad3 inhibited the up-regulatory effect of TGF-beta, and reduced the basal expression of MMP-13 mRNAs by 70%. In addition, dominant negative Smad3 inhibited the up-regulatory effect of TGF-beta on collagenase-1 (MMP-1) mRNA levels by 60%. The overexpression of Smad2 had no effect on MMP-13 or MMP-1 mRNA expression. In addition, adenoviral expression of dominant negative Smad3 inhibited invasion of UT-SCC-7 cells through Matrigel. Together, these results provide evidence for a crucial role of Smad3 in the invasive phenotype of human SCC cells, and suggest Smad3 as a target to specifically inhibit their invasion. The results also show, that the activation of SCC cell invasion by TGF-beta is uncoupled from its inhibitory effect on cell proliferation.